The long-term goals of this project are to advance our knowledge about the functions of H1 linker histones and to understand the functional significance of the diversity present in this family of chromatin proteins. In most eukaryotic cells H1 linker histones are nearly as abundant as nucleosome core particles. Therefore, H1 histones play a key role in the structure of the chromatin fiber. H1 histones affect gene expression as well as many other processes requiring access to DNA. Much of our knowledge about the functions of H1 has been derived from in vitro experiments. Our approach is to analyze the functions of H1 histones in vivo in mice and in embryonic stem (ES) cells. The mouse (and other mammalian) H1 histones consist of at least 8 nonallelic variants or subtypes that differ considerably in their primary sequences and in their expression during development and tissue differentiation. These subtypes offer an additional level of regulation of chromatin function. Our strategy for studying the functions of linker histones has been to generate and characterize mice and ES cell lines in which one or more H1 genes have been inactivated by gene targeting. We have generated a large collection of mouse strains consisting of both single null mutants (6 of the 8 subtypes have been inactivated) and compound null strains. One of our most informative strains has 3 H1 genes inactivated simultaneously (triple knock-out, TKO). This mutant demonstrated that, unlike in less complex eukaryotes, H1 histones are essential for mammalian development. Some of the mutant strains and TKO ES cells have been analyzed for effects on gene expression. The results showed that, contrary to conclusions derived from in vitro experiments, H1 is not a global repressor of transcription. Importantly, we discovered that H1 is involved in promoting DNA methylation at imprinted gene loci. We now propose to use our unique set of mouse strains and ES cell lines to: (1) Understand the mechanism(s) by which H1 controls DNA methylation. We also propose to identify on a genome-wide scale the sites in the genome at which H1 affects DNA methylation. (2) Assess the role of H1 posttranslational modifications and H1 subtype diversity in the ability of H1 to regulate gene expression and DNA methylation. (3) Develop new mutant mouse strains and ES cell lines, specifically a conditional triple H1 null, and use them to study the role of H1 in postnatal development, cell differentiation and tissue-specific gene expression. There is evidence that H1 histones are downstream targets of cyclin-dependent kinases regulated by the retinoblastoma protein. Thus, H1 histones may be key transducers of information between cell cycle regulators and chromatin structure, gene expression and other activities of the genome in normal and malignant cells. We think we have a unique opportunity to help understand some of the numerous important functions of this major component of eukaryotic chromosomes.